Bone substitute material for medical use and method of producing the same

ABSTRACT

There is disclosed a bone substitute material for medical use which satisfies all the requirements of (1) no histotoxicity, (2) osteoconductivity, (3) bone replacement capability, and (4) mechanical strength necessary for a bone reconstruction operation. The bone substitute material for medical use is predominantly composed of carbonate apatite and produced through the formation of carbonate apatite by contacting a block of calcium compound with a phosphate-containing solution, wherein the calcium compound block contains substantially no powders, and at least one of said calcium compound block and said phosphate solution contains a carbonate group, without any sintering. The block of calcium compound is preferably one prepared using an artificially synthesized calcium compound, most preferably a foamed calcium compound.

TECHNICAL FIELD

The present invention relates to a hone substitute material for medicaluse and a method of producing the same, and more particularly, to a bonesubstitute material for use in the regeneration or repair of the hardtissue.

BACKGROUND ART

In medical and dental fields, there are many cases where a bone defectcaused by a disease or injury must be repaired or regenerated. The firstpossible approach to repair the bone defect may be an autograft bonetransplantation, but this approach causes problems including an invasioninto the sound tissue owing to the autograft bone as well asquantitative and morphological limitation of the collected bone.Artificially produced bone substitute materials are therefore clinicallyemployed.

It is desired for a bone substitute material to combine the features of(1) no histotoxicity, (2) osteoconductivity, (3) bone replacementcapability, and (4) mechanical strength required in a bonereconstruction operation. No histotoxicity is indispensable to abiomaterial. Histotoxicity is evaluated from macroscopical orhistopathological indication of inflammation in an experimental animalimplanted with a bone substitute material. Histotoxicity is alsoinfluenced by the mechanical strength of a bone substitute material.There occurs crystalline inflammation if the mechanical strength of thebone substitute material is low so that the material is partlydisintegrated during the bone reconstruction into powdery form andremains within the bone defect. Osteoconductivity is defined as theproperty of a bone substitute material applied to a bone defect topromote the formation of new bone tissue from the site of application tothe bone so as to cover the bone substitute material. Osteoconductivityis a crucial property of a bone substitute material, the presence orabsence of which is evaluated on histopathological examination of anexperimental animal implanted with the bone substitute material. It isgenerally considered that if the differentiation of osteoblast cellscultured on a bone substitute material is promoted, then the materialpossesses osteoconductivity.

Bone replacement capability is crucial to a bone substitute material. Inthe bone replacement by the bone substitute material, it is ideal thatthe resorbing process by osteoclast cells and the bone formation processby osteoblast cells proceed as in the remodeling. Replacement of bone bythe bone substitute material is histopathologically evaluated with anexperimental animal implanted with the bone substitute material.However, the evaluation of the bone replacement capability with suchexperimental animals requires much time for the experiments. Anosteoconductive material operates on the principle of replacing bone asresorption by osteoclast cells proceds. The possible bone replacementcapability of such a material can therefore be ascertained by checkingresorption cavities formed by the osteoclast cells on the material. Itis also crucial for a bone substitute material to have the mechanicalstrength necessary for a bone repair operation. While the degree of therequired mechanical strength is not necessarily definite, it is ofcourse indispensable for the material to possess a mechanical strengthresistant to the implantation.

Hydroxyapatite is currently the most-studied bone substitute material.The primary inorganic component of the bone, tooth or other hard tissueof verterbrates including humans is an apatite basically composed ofhydroxyapatite, Ca₁₀(PO₄)₆(OH)₂. Thus, there is clinically used a bonesubstitute material composed of sintered hydroxyapatite prepared bysintering chemically synthesized hydroxyapatite. While sinteredhydroxyapatite exhibits osteoconductivity and therefore is a very usefulbone substitute material, it is an unresorbable material which will notbe resorbed at bone defect sites even over the passage of time. Boneexhibits biological functions such as hematopoiesis, and it is ideal touse a bone substitute material which is capable of replacing bone.

Under the circumstances are also clinically used as bone substitutematerial such materials as β-tricalcium phosphate, calcium sulfate,calcium carbonate and the like. These materials exhibit resorbabilitybut are not osteoconductive, or are less osteoconductive thanhydroxyapatite. In addition, when there is used a resorbing materialsuch as β-tricalcium phosphate, calcium sulfate or calcium carbonate,the resorption is caused by physicochemical dissolution or extraneousgiant cells and the mechanism of the resorption is not linked with boneformation by osteoblast cells. Thus, in a case where bone defect issevere or the bone defect site is inferior in bone formation abilitybecause of aging and other reasons, the bone resorption proceeds evenbefore the bone formation is sufficiently carried out, resulting in thebone substitute material being consumed prior to its replacement ofbone, and the bone defect comes to be repaired by fibrous connectivetissue.

In the case of an autogenous bone transplantation, the mechanism of bonereplacement by the transplanted bone is the same as that of theremodeling of living bone. Thus, bone resorption is advanced byosteoclast cells while bone formation is accomplished by osteoblastcells. In sintered hydroxyapatite which exhibits osteoconductivity,although the process of bone formation by osteoblast cells proceeds, nobone replacement occurs because bone is not resorbed by osteoclastcells. Resorption by osteoclasts is accomplished through the formationof Howship's lacunae, in the interior of which there is induced a low pHresulting in the decomposition of bone apatite. Bone apatite iscarbonate apatite containing a carbonate group and thereforedecomposable in the low pH environment induced by osteoclast cells. Bycontrast, since sintered hydroxyapatite contains no carbonate group, itis not decomposed in the low pH environment induced by osteoclast cells.Thus, sintered hydroxyapatie, which is currently put into clinical useas a bone substitute material, has no bone replacement capability partlybecause it is not a carbonate-containing apatite.

In view of there considerations, carbonate apatite would be an idealbone substitute material. However, no technology for producing carbonateapatite practically utilizable as a bone substitute material has notbeen established. More specifically, known or proposed uses of carbonateapatite are limited to those as adsorbents or carriers for biomaterialsand the like, and as restorative materials for bones and teeth [forexample, Japanese Patent Application Publication No. 1995-61861 (PatentReference No. 1), Japanese Patent Application Publication No. 1998-36106(Patent Reference No. 2), Japanese Patent Application Publication No.1999-180705 (Patent Reference No. 3)] The latter use is only for thepurpose of filling the defect sites of bones or teeth, and no materialprimarily composed of carbonate apatite has been developed whichsatisfies the prerequisites of a bone substitute material for medicaluse, including bone replacement capability and no histotoxicity withoutcausing inflammation.

-   Patent Reference No. 1: Japanese Patent Application Publication No.    1995-61861-   Patent Reference No. 2: Japanese Patent Application Publication No.    1998-36106-   Patent Reference No. 3: Japanese Patent Application No. 1999-180705

DISCLOSURE OF THE INVENTION Subjects to be Solved by the Invention

The object of the present invention is to provide a bone substitutematerial for medical use which satisfies all the requirements of (1) nohistotoxicity, (2) osteoconductivity, (3) bone replacement capability,and (4) mechanical strength necessary for a bone reconstructionoperation.

Through extensive studies, the present inventors discovered that therecan be obtained a bone substitute material for medical use satisfyingall the above-mentioned requirements by forming carbonate apatite from acalcium compound and a phosphate solution (an aqueous solution) in thepresence of carbonate functional group under a specific condition,thereby achieving the present invention.

Thus, according to the present invention there is provided a method ofproducing a bone substitute material predominantly composed of carbonateapatite for medical use, which comprises the step of forming carbonateapatite by contacting a block of calcium compound with aphosphate-containing solution, wherein said calcium compound blockcontains substantially no powders, wherein at least one of said calciumcompound block and said phosphate solution contains a carbonate group,and wherein the method does not include any sintering step.

In a preferred embodiment of the present invention, the block of calciumcompound is one prepared using an artificially synthesized calciumcompound, most preferably a foamed calcium compound.

The present invention also provides a bone substitute materialpredominantly composed of carbonate apatite for medical use, produced bythe above-mentioned method, which contains carbonate group in an amountof 0.5% or more by weight.

Advantageous Effects of the Invention

The present invention provides a bone substitute material for medicaluse which satisfies all the requirements of (1) no histotoxicity, (2)osteoconductivity, (3) bone replacement capability, and (4) mechanicalstrength necessary for a bone reconstruction operation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a powder X-ray diffraction pattern of the bonesubstitute material for medical use produced in Example 1.

FIG. 2 illustrates a Fourier-transformed infrared spectrum of the bonesubstitute material for medical use produced in Example 1.

FIG. 3 illustrates a powder X-ray diffraction pattern of the bonesubstitute material for medical use produced in Example 3.

FIG. 4 illustrates a Fourier-transformed infrared spectrum of the bonesubstitute material for medical use produced in Example 3.

FIG. 5 illustrates a powder X-ray diffraction pattern of the bonesubstitute material for medical use produced in Comparative Example 2.

FIG. 6 illustrates a Fourier-transformed infrared spectrum of the bonesubstitute material for medical use produced in Comparative Example 2.

FIG. 7 shows an electron microscope photograph of the bone substitutematerial produced in Example 4.

FIG. 8 illustrates a powder X-ray diffraction pattern of the bonesubstitute material for medical use produced in Example 4.

FIG. 9 illustrates a Fourier-transformed infrared spectrum of the bonesubstitute material for medical use produced in Example 4.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is directed to the formation of carbonate apatiteby contacting a calcium compound and a phosphate solution in thepresence of carbonate group, which is characterized by thebelow-mentioned matters (I) to (III), thereby realizing for the firsttime a bone substitute material for medical use satisfying all therequirements of (1) no histotoxicity, (2) osteoconductivity, (3) bonereplacement capability, and (4) mechanical strength necessary for a boneprosthesis operation.

(I) The first characteristic feature of the method of producing a bonesubstitute material for medical use according to the present inventionresides in the utilization of a block of calcium compound containingsubstantially no powders, as the starting calcium compound. As usedherein, the term “block” refers to a substance which is in solid statebut is not powdery, as exemplified by granule, solid and porous bodies.Thus, the block has a number-averaged particle diameter (particle size)of preferably 0.2 mm or larger, most preferably 0.5 mm or larger, andthe most preferably and completely satisfactorily 1 mm or larger. Aparticularly preferred example of calcium compound for use in thepresent invention is a foamed calcium compound. As used herein withrespect to the present invention, by the phrase “containingsubstantially no powders” is meant that the block as mentioned abovecontains powders with a diameter of 20 μm or smaller in an amount ofless than 1% by weight.

A surprising discovery of the present inventors is that the utilizationof such “a block of calcium compound containing substantially nopowders” can produce a bone substitute material having no histotoxicitywithout causing inflammatory reaction. This is possibly because, onimplanting the bone substitute material, phagocytes such as exogenousgiant cells englobe carbonate apatite powder particles of a small size,thereby not inducing the inflammatory response. In terms ofhistocompatibility, the powders with a diameter of 20 μm or smallerwhich may be contained in the block material is preferably less than0.8% by weight, and more preferably less than 0.5% by weight. Bycontrast, in the conventional uses of carbonate apatite such as inadsorbents, the starting calcium compound is in powdery state. Forexample, in Japanese Patent Application Publication No. 1998-36106(Patent Reference No. 2) it is required that calcium carbonate as theraw material should have an average particle diameter in the range of 1to 50 μm. Calcium carbonate from such powdery starting material willnever produce a practical bone substitute material to which the presentinvention is directed.

(II) The second characteristic feature of the method of producing a boneprosthesis for medical use according to the present invention is that itonly comprises the step of forming carbonate apatite by contacting acalcium compound, as explained in the above (I), with a phosphatesolution, under the condition where at least one of the calcium compoundand the phosphate solution contains carbonate group, without anysubsequent step of sintering.

In a reaction system to which the present invention is directed, whereincarbonate apatite is thermodynamically stable as compared with carbonatecalcium, there is formed carbonate apatite with a sufficient hardness,even at a low temperature at which sintering will not take place.Conversely, sintering will not produce the desired carbonate apatitebecause carbonate group is irreversibly removed during the sinteringstep. In addition, sintering will advance the crystallinity whichdecreases the specific surface area, resulting in difficulty inresorption by osteoclast cells as well as no or extremely slow bonereplacement. Thus, sintered apatite carbonate as conventionally proposed(for example, the one as described in Japanese Patent ApplicationPublication No. 1995-61861, Patent Reference No. 1, in which powderyapatite carbonate is sintered at a temperature of 600 to 850° C.) is noteffective for use in a bone substitute material for medical use of thepresent invention having an excellent bone replacement capability.

(III) In order to obtain a bone substitute material for medical useaccording to the invention which satisfies all the above-mentionedrequirements, it is necessary to take into consideration the purity of acalcium compound as the starting material, in addition to the aforesaidmatters (I) and (II).

The leading source of histotoxicity causing inflammation in a boneprosthesis is impurities or antigenic substances contained in thestarting calcium compound. In this respect, as the starting calciummaterial for use in the present invention is preferred an artificiallysynthesized calcium compound since it is intrinsically low inimpurities. By contrast, it has been hitherto proposed that carbonateapatite be produced through the fragmentation of naturally occurringcalcium compounds. For example, in Japanese Patent ApplicationPublication No. 1999-180705 (Patent Reference No. 3), there are usedpulverized limestones or the like as the starting material of apatitecarbonate apatite for use in adsorbents or carriers of biomaterials.However, such naturally occurring materials cannot produce a bonesubstitute material of the present invention which does not causeinflammation due to histotoxicity, because they contain inherentlynatural impurities in addition to the unfavorable powders (those havinga diameter of 20 μm or smaller) as explained in the above (I).

Embodiments of the present invention will be detailed below withreference to the constitutional elements of the bone substitute materialand the method of producing the same according to the present invention.

As used herein with respect to the present invention, “an artificiallysynthesized calcium compound” refers to a calcium compound which hasbeen synthesized by a chemical method or the like, such as a reagent. Anaturally occurring or organism-derived calcium compound is not “theartificially synthesized” material. For example, while natural gypsumpowders or bone meals are calcium compounds, they are not artificiallysynthesized calcium compounds. However, a calcium compound which hasbeen produced by processing natural gypsum, bone or the like via, forexample, a dissolution-refining process is an artificially synthesizedcompound.

As used herein with respect to the present invention, “a calciumcompound” refers to a calcium-containing compound, exemplified by metalcalcium, calcium hydroxide, calcium carbonate, calcium chloride, calciumacetate, calcium benzoate, calcium fluoride, calcium formate, calciumgluconate, calcium hydride, calcium iodide, calcium calcium lactate,apatite, tricalcium phosphate, tetracalcium phosphate, calciumhydrogenphosphate, calcium silicate, and the like. A single calciumcompound can be used, while a mixture of a plurality of calciumcompounds can also be used.

As used herein with respect to the present invention, “a phosphate”refers to a phosphate group-containing compound, exemplified byphosphoric acid, triammonium phosphate, tripotassium phosphate,trisodium phosphate, disodium ammoniumphosphate, sodiumdiammoniumphosphate, ammonium dihydrogenphosphate, potassiumdihydrogenphosphate, sodium dihydrogenphosphate, trimagnesiumphosphate,ammonium sodium hydrogenphosphate, diammonium hydrogenphosphate,dipotassium hydrogenphosphate, disodium hydrogenphosphate, magnesiumhydrogenphosphate-tridiacetyl phosphate, diphenyl phosphate, dimethylphosphate, cellulose phosphate, ferrous phosphate, ferric phosphate,tetrabutylammonium phosphate, copper phosphate, triethyl phosphate,tricresyl phosphate, tris-trimethylsilyl phosphate, triphenyl phosphate,tributyl phosphate, trimethyl phosphate, guanidine phosphate, cobaltphosphate, and the like. A single phosphate-containing compound can beused, while a mixture of a plurality of phosphate-containing compoundscan also be used.

In the present invention, use of a carbonate group-containing compoundensures that at least one of the calsium compound block and thephosphate solution contains phosphate group(s). As used herein, “acarbonate group-containing compound” refers to carbon dioxide (CO₂) or acompound of carbonate group (CO₃ ²⁻) and cation, exemplified by carbondioxide gas, dry ice, sodium hydrogencarbonate, disodium carbonate,potassium hydrogencarbonate, dipotassium carbonate, ammoniumhydrogencarbonate, diammonium carbonate, calcium carbonate, and thelike. A single carbonate group-containing can be used, while a mixtureof a plurality of carbonate group-containing compounds can also be used.

As used herein with respect to the present invention, “apatite” refersto a compound having a basic structure expressed by the formulaA₁₀(BO₄)₆C₆, wherein A denotes Ca²⁺, Cd²⁺, Sr²⁺, Ba²⁺, Pb²⁺, Zn²⁺, Mg²⁺,Mn²⁺, Fe²⁺, H⁺, H₃O⁺, Na⁺, K⁺, Al³⁺, Y³⁺, Ce³⁺, Nd³⁺, La³⁺, C⁴⁺ or thelike, BO₄ denotes PO₄ ³⁻, CO₃ ²⁻, CrO₄ ³⁻, AsO₄ ³⁻, VO₄ ³⁻, UO₄ ³⁻, SO₄²⁻, SiO₄ ⁴⁻, GeO₄ ⁴⁻ or the like, C denotes OH⁻, OD⁻, F⁻, Br⁻, BO²⁻, CO₃²⁻, O²⁻, or the like. As used herein with respect to the presentinvention, “hydroxyapatite” is Ca₁₀(PO₄)₆(OH)₂. As used herein withrespect to the present invention, “carbonate apatite (apatitecarbonate)” refers to an apatite in which a part or all of phosphategroups or hydroxyl groups therein are replaced with carbonate groups.The apatite in which phosphate groups are replaced with carbonate groupsare called B-type carbonate apatite, while the apatite in which hydroxylgroups are replaced with carbonate groups are called A-type carbonateapatite.

As used herein with respect to the present invention, “foamed orfoamed-shaped” refers to a three dimensional structure with continuouspores (the so-called sponge) such as in polyurethane foam.

A calcium compound block for use in the present invention can beproduced, for example, by the calcinations of an artificiallysynthesized calcium compound, the hardening reaction of an air-hardeningcement or the hardening reaction of a hydraulic cement.

The production of a calcium compound block by the calcination of acalcium compound may be carried out, for example, as follows: Tricalciumphosphate powders are compression-molded uniaxially, followed bycalcining the resultant at 1500° C. for six hours, so as to producetricalcium phosphate block. In the present invention, the production ofsuch calcium phosphate block is for the purpose of providing a calciumcomponent for a bone substitute material for medical use which ispredominantly composed carbonate apatite, and therefore there is no needof a special device such as HIP or CIP for the prevention of theelimination of carbonate group, although the use of such device is notexactly excluded. Thus, since there is no need for HIP or CIP, the costof production is low, making mass production possible, while there canbe produced a bone substitute material in a desired shape.

The production of a calcium compound block by the hardening reaction ofan air-hardening cement is exemplified by the reaction of calciumhydroxide and carbon dioxide: Calcium hydroxide is compression-moldeduniaxially, and then the resultant compressed body is rendered to reactwith carbon dioxide under a stream of steam, thereby producing calciumcarbonate block as precipitate, in which the compressed calciumhydroxide is partly or wholly replaced by calcium carbonate.

The production of a calcium compound block by the hardening reaction ofa hydraulic cement is exemplified by the use of gypsum: Gypsum iskneaded with water, and the resultant is poured into a desired moldwhere the gypsum hardens to produce calcium sulfate block. Theproduction of such calcium compound block from a hydraulic cement suchas gypsum is advantageous in that it enables the production of a calciumcompound block in a desired shape in an easy manner.

Calcium compound block occurs even naturally in abundance, for example,as coral, marble, natural gypsum or the like. However as mentionedpreviously, these naturally occurring calcium compounds are not suitableas a biomaterial because of impurities contained, and therefore in theproduction of the calcium compound for use in the present inventionthere must be used a highly pure starting material which has beenartificially synthesized through a chemical synthesis or the like.Likewise, organism-derived materials are not suitable as the biomaterialbecause of antigenicity.

While there is no specific restriction on the shape of the calciumcompound block, a porous shape is preferred since it is advantageous inaccelerating bone replacement rate. In the case of a porous body, theporosity is preferably 10% or over, more preferably 30% or over, and themost preferably 50% or over. Particularly preferred calcium compoundblock is a foamed-shaped one because it provides a bone substitutematerial with an accelerated bone replacement. A foamed calcium compoundblock can be produced by a known method, for example, by the method asdescribed in “Development of Porous Ceramics” (Edited by S. Hattori andA. Yamanaka, Published by CMC Co. Ltd., Pages 277-294 (1991)). Morespecifically, cell membrane-removed soft polyurethane foam is immersedin a calcium compound suspension, thereby rendering the calcium compoundadhered on the surface of the trabecula of the polyurethane foam. Driedcalcium compound-adhered polyurethane foam is calcined at a desiredtemperature to sinter the calcium compound while burning out thepolyurethane foam, thereby producing the foamed calcium compound.

A continuous pore foamed structure is quite useful in the method ofproducing a bone substitute material according to the present invention,in which a foamed calcium compound block is used so that bone cellspenetrate into the interior of the block effecting three-dimensionalbone replacement. In view of the penetration of bone cells, the foamedcalcium compound block has an average pore diameter preferably in therange of 50 μm to 1000 μm, more preferably in the range of 100 μm to 500μm, and the most preferably in the range of 200 μm to 300 μm.

The calcium compound block produced in the above-mentioned manner isthen brought into to contact with a solution containing a phosphate. Thephosphate solution generally has a pH of 4.5 or higher. The contact iscarried out, for example, by immersing the calcium compound in thesolution or by spraying the solution upon the calcium compound. Ingeneral the immersion is the most convenient and economical.

In contacting a calcium compound block with a phosphate-containingsolution, in the case where the calcium compound block is of acomposition not containing carbonate group, it is indispensable to add acarbonate group-containing component to the phosphate-containingsolution. In the case of a carbonate group-containing calcium compoundblock such as calcium carbonate block, it is not necessarily requiredfor the phosphate-containing solution to contain carbonate group.However, it is quite acceptable for the phosphate-containing solutionalso to contain carbonate group for the purpose of adjusting thecarbonate group content of the bone substitute material to be produced.A carbonate group-containing component may be dissolved in thephosphate-containing solution or may be added thereto even in an amountexceeding saturation. In any case, it is required that the carbonategroup content be sufficient for the bone substitute material to beproduced.

While there is no restriction on the temperature at which a calciumcompound block is contacted with a phosphate-containing solution, thehigher is the temperature, the more rapidly proceeds the production ofthe bone substitute material for medical use predominantly composed ofcarbonate apatite. Thus, the reaction temperature is preferably 50° C.or higher, and more preferably 80° C. or higher. Hydrothermal synthesisat 100° C. or higher is particularly preferred because the carbonateapatite formation proceeds even internally and the production rate isaccelerated as well. However, as described previously, no sintering stepis required in the method of the production of a bone substitutematerial according to the present invention.

There are no restrictions on the period of time for which the calciumcompound block is contacted with the phosphate-containing solution,either. Such period of time for the contact can be determined as deemedappropriate depending upon the composition of the bone substitutematerial for medical use to be produced.

The mechanism has not yet been completely elucidated by which a bonesubstitute material for medical use, predominantly composed of carbonateapatite, can be produced in accordance with the method of the presentinvention. It is however considered, as discussed previously, that in acase where carbonate group is present, carbonate apatite assumesthermodynamically the most stable phase, and therefore the calciumcompound is converted to carbonate apatite.

While no particular restrictions are imposed on the carbonate apatitecontent in the bone substitute material produced, it is preferred, inview of the bone replacement rate and other factors, that carbonateapatite content in the composition be 50% or more by weight, and morepreferably 70% by weight in the composition. It is particularlypreferred that carbonate apatite is contained in 90% or more by weightin the composition.

The carbonate group content in the bone substitute material producedexerts a large influence on the bone replacement rate. As the carbonategroup content increases, the bone replacement rate accelerates. Thepresent invention enables the production of carbonate apatite having acarbonate group content of 0.5% or more by weight with a highlyincreased bone replacement rate as compared with the conventionallyknown sintered hydroxyapatite. The carbonate group content in the bonesubstitute material is preferably 2% or more by weight, more preferably4% or more by weight and the most preferably 6% or more by weight.

While the present invention will be explained in more detail withreference to Examples and Comparative Examples given below, the presentinvention is not limited to such Examples.

With respect to powders having a particle diameter of 20 μm or smallercontained in the carbonate apatite blocks prepared in the Examples andthe Comparative Examples, the percentage of powder content wasdetermined as follows: Carbonate apatite blocks weighing about 10 g wereimmersed in 200 ml of distilled water, followed by stirring carefullywith a stirring rod so that the carbonate apatite blocks did not collidewith the vessel or the stirring rod. Then, the total distilled waterwith the carbonate apatite blocks immersed therein was sifted through a140-mesh sieve, followed by flushing the carbonate apatite blocksstanding on the 140-mesh sieve with 100 ml water. This operation wasrepeated three times. The distilled water and the powders sifted throughthe 140-mesh sieve were then filtered through six different filterpapers as standardized by JIS P3801, followed by drying and weighingoperations. The dried powders were suspended in distilled water toanalyze pore size distribution using a sedimentation particle sizeanalyzer. Thus, with respect to powders having a particle size (particlediameter) of 20 μm or smaller contained in carbonate apatite, the powdercontent was determined by the percentage of such powders in thedistribution as well as the total weight of the carbonate apatite siftedthrough the 140-mesh sieve.

Example 1

Hemihydrated calcium sulfate (Nacalai Tesque Inc.) was kneaded withdistilled water at a ratio of the water of 0.4 (ml/g) to produce calciumsulfate blocks. The thus prepared calcium sulfate was immersed in a 1Mdiammonium hydrogenphosphate solution (in which ammonium carbonate hadbeen suspended) for two days at 80° C.

The X-ray diffraction pattern (FIG. 1) and the Fourier-transformedinfrared spectrum (FIG. 2) of the block product showed that it wascomposed of carbonate apatite. It was also found by measurement with aCHN analyzer that the carbonate group content was 7% by weight. Theindirect tensile strength of the bone substitute material produced wasfound to be 1.2 MPa. With respect to the powders having a particle sizeof 20 μm or smaller contained in the bone substitute material produced,the powder content was approx. 0.002% by weight.

The carbonate apatite blocks produced were inoculated with osteoblastcells, and cultivation was carried out. The osteocalcine value, acriterion for osteoconductivity, was 11 ng/ml on 15th day and markedlyhigher than the value of cell culture dish, 3 ng/ml, demonstrating thatthe product had osteoconductivity. The cultivation of osteoclast cellson the carbonate apatite blocks resulted in the formation of resorptioncavities, suggesting that the bone substitute material had bonereplacement capability. There was no indication of inflammation when theprosthesis material was implanted into a rat tibia. It was alsorecognized from the histopathological observation that there was inducedosteoconduction with the carbonate apatite block produced.

Example 2

A mixture of an equal amount by weight of hemihydrated calcium sulfate(Nacalai Tesqui Inc.) and calcium carbonate (Nacalai Tesque Inc.) waskneaded with distilled water at a ratio of the water of 0.4 (ml/g) toproduce calcium blocks. The thus produced calcium sulfate blocks wereimmersed in a 1M diammonium hydrogenphosphate solution (in whichammonium carbonate had been suspended) for two days at 80° C.

The X-ray diffraction pattern and the Fourier-transformed infraredspectrum of the block product showed that it was composed of carbonateapatite. It was also found by measurement with a CHN analyzer that thecarbonate group content was 8% by weight. The indirect tensile strengthof the bone substitute material produced was found to be 4.8 MPa. Withrespect to the powders having a particle size of 20 μm or smallercontained in the carbonate apatite block produced, the powder contentwas approx. 0.005% by weight.

The carbonate apatite blocks produced were inoculated with osteoblastcells, and cultivation was carried out. The osteocalcine value, acriterion for osteoconductivity, was 12 ng/ml on 15th day and markedlyhigher than the value of cell culture dish, 3 ng/ml, demonstrating thatthe product had osteoconductivity. The cultivation of osteoclast cellson the carbonate apatite blocks resulted in the formation of resorptioncavities, suggesting that the bone substitute material produced has bonereplacement capability. There was no indication of inflammation when theprosthesis material was implanted into a rat tibia. It was alsorecognized from the histopathological observation that there was inducedosteoconduction with the carbonate apatite block produced.

Comparative Example 1

This example relates to the production of apatite block not containingcarbonate group, in order to demonstrate the advantageous effects of thepresent invention.

Calcium sulfate (Nacalai Tesque Inc.) was kneaded with distilled waterat a ratio of the water of 0.4 (ml/g) to produce calcium sulfate blocks.The thus produced calcium sulfate blocks were immersed in a 1Mdiammonium hydrogenphosphate solution (which had been nitrogen-replaced)for two days at 80° C.

The X-ray diffraction pattern and the Fourier-transformed infraredspectrum of the block product showed that it was composed of apatite notcontaining carbonate group. The indirect tensile strength of the bonesubstitute material produced was found to be 1.8 MPa. With respect tothe powders having a particle size of 20 μm or smaller contained in theapatite block produced, the powder content was approx. 0.005% by weight.

The apatite blocks produced were inoculated with osteoblast cells, andcultivation was carried out. The osteocalcine value, a criterion forosteoconductivity, was 8 ng/ml on 15th day and markedly higher than thatof cell culture dish, 3 ng/ml, showing that the product hadosteoconductivity, but the value was lower than those with the bonesubstitute materials produced in Examples 1 and 2. No resorptioncavities by osteoclast cells were observed, indicating that there was nobone replacement with the bone substitute material of this ComparativeExample 1, which falls outside the scope of the present invention. Therewas no indication of inflammation when the prosthesis material wasimplanted into a rat tibia. It was also recognized from thehistopathological observation that there was induced osteoconductionwith the apatite block produced.

Example 3

Calcium hydroxide (Nacalai Tesque Inc.) 0.2 g was compression-molded ina circular mold at an axial compression pressure of 20 kg/cm² and theresultant compressed body was subjected to carbonation under a stream ofcarbon dioxide with a relative humidity of 100% to produce calciumcarbonate blocks. The X-ray diffraction pattern and theFourier-transformed infrared spectrum of the block product showed thatit was composed of calcium carbonate. The calcium carbonate blocks wereimmersed in a 1M disodium hydrogenphosphate solution at 60° C. for sevendays, produce blocks in the same shape with the calcium carbonate block.

The X-ray diffraction pattern (FIG. 3) and the Fourier-transformedinfrared spectrum (FIG. 4) of the block product showed that it wascomposed of carbonate apatite. It was also found by measurement with aCNN analyzer that the carbonate group content was 11% by weight. Theindirect tensile strength of the bone substitute material produced wasfound to be 1.8 MPa. With respect to the powders having a particle sizeof 20 μm or smaller contained in the carbonate apatite block produced,the powder content was approx. 0.005% by weight.

The carbonate apatite blocks produced were inoculated with osteoblastcells, and cultivation was carried out. The osteocalcine value, acriterion for osteoconductivity, was 13 ng/ml on 15th day and markedlyhigher than the value of cell culture dish, 3 ng/ml, demonstrating thatthe product had osteoconductivity. The cultivation of osteoclast cellsresulted in the formation of resorption cavities, suggesting that thebone substitute material produced had bone replacement capability. Therewas no indication of inflammation when the prosthesis material wasimplanted into a rat tibia. It was also recognized from thehistopathological observation that there was induced osteoconductionwith the carbonate apatite block produced.

Comparative Example 2

This example relates to the production of sintered hydroxyapatite whichdoes not contain carbonate group, in order to demonstrate theadvantageous effects of the present invention.

Hydroxyapatite powders (Taihei Chemicals Ltd.) were compression-moldedat an axial pressure of 20 kg/cm². The resultant compressed body washeated up to 1250° C. at the rate of 4° C. per minute and maintained at1250° C. for six hours, followed by cooling to produce sinteredhydroxyapatite.

The X-ray diffraction pattern (FIG. 5) and the Fourier-transformedinfrared spectrum (FIG. 6) of the sintered hydroxyapatite body producedshowed that it was composed of apatite not containing carbonate group.The indirect tensile strength of the bone substitute material producedwas found to be 90 MPa. With respect to the powders having a particlesize of 20 μm or smaller contained in the apatite block produced, thepowder content was approx. 0.001% by weight.

The apatite blocks produced were inoculated with osteoblast cells, andcultivation was carried out. The osteocalcine value, a criterion forosteoconductivity, was carried out. The osteocalcine value, a criterionfor osteoconductivity, was 8 ng/ml on 15th day and markedly higher thanthat of cell culture dish, 3 ng/ml, showing that the product hadosteoconductivity but the value was lower than those with the bonesubstitute materials produced in Example 1, 2 and 3. No resorptioncavities by osteoclast cells were observed, indicating that there was nobone replacement with the bone substitute material produced in thisComparative Example 2, which falls outside the scope of the presentinvention. There was no indication of inflammation when the prosthesismaterial was implanted into a rat tibia. It was also recognized from thehistopathological observation that there was induced osteoconductionwith the apatite block produced.

Comparative Example 3

This example relates to the production of apatite block from a naturallyoccurring material, in order to demonstrate the advantageous effects ofthe present invention. Ground natural limestones having a particle sizeof about 1 mm immersed in a 1M disodium hydrogenphosphate solution at60° C. for seven days to produce blocks in the same shape with thestarting ground limestones.

The X-ray diffraction pattern and the Fourier-transformed infraredspectrum of the block product showed that it was composed of carbonateapatite. It was also found by measurement with a CHN analyzer that thecarbonate group content was 10% by weight. With respect to the powdershaving a particle size of 20 μm or smaller contained in the carbonateapatite block produced, the powder content was approx 1.2% by weight. Onimplanting the product into a rat tibia, there was observed clearindication of inflammation presumably due to impurities inherentlycontained in the naturally occurring material. From the histologicalobservation there was recognized no osteoconduction with the carbonateapatite block product.

Comparative Example 3-2

This example relates to the production of carbonate apatite blockfalling outside the scope of the present invention, in order todemonstrate the advantageous effects of the present invention,particularly in terms of the state of the powder. For the purpose ofremoving powders having a particle size of 20 μm or smaller contained inthe carbonate apatite blocks as produced in Comparative Example 3, thecarbonate apatite blocks produced were placed on a 140-mesh sieve,followed by flushing with water no that fine powders passed through thesieve. The carbonate apatite blocks remaining on the sieve were dried.The thus produced carbonate apatite block had a powder content ofapproximately 0.05% by weight with respect to the powders having aparticle size of 20 μm or smaller.

On implanting the product into a rat tibia, there was observed a clearindication of inflammation presumably due to impurities inherentlycontained in the naturally occurring material. While the inflammationwas mild as compared with that in Comparative Example 3, from thehistological observation there was recognized no osteoconduction withthe carbonate apatite product here again.

Example 4

α-tricalcium phosphate powders (Taihei Chemicals Ltd.) were admixed withwater at a ratio of 1:1 by weight to prepare a suspension. Polyurethanefoam (Bridgestone Corp.) was immersed in the suspension of α-tricalciumphosphate powders, followed by drying. The thus prepared polyurethanefoam with the α-tricalcium phosphate powders adhered to the trabeculaethereof was calcined at 1500° C. for five hours by raising thetemperature at the rate of 1° C. per minute up to 400° C. and then atthe rate of 5° C., whereafter the calcined product was cooed. Thus,there was produced sintered α-tricalcium phosphate foam.

The α-tricalcium phosphate foam produced was immersed in an aqueoussolution in which are suspended sodium carbonate and disodium hydrogenphosphate, to undergo hydrothermal treatment at 200° C. for twelvehours.

FIG. 7 shows the foamed compound obtained. The X-ray diffraction pattern(FIG. 8) and the Fourier-transformed infrared spectrum (FIG. 9) of thefoamed compound showed that it was composed of carbonate apatite. It wasalso found by measurement with a CHN analyzer that the carbonate groupcontent was 6% by weight. The indirect tensile strength was found to be0.3 MPa. With respect to the powders having a particle size of 20 μm orsmaller contained in carbonate apatite block produced, the power contentwas approx 0.001% by weight.

The carbonate apatite blocks produced were inoculated with osteoblastcells, and cultivation was carried out. The osteocalcine value, acriterion for osteoconductivity, was 13 ng/ml on 15th day and markedlyhigher than the value of cell culture dish, 3 ng/ml, demonstrating thatthe product had osteoconductivity. The formation of resorption cavitiesby osteoclast cells indicated that the bone substitute material producedhad bone replacement capability. There was no indication of inflammationwhen the prosthesis material was implanted into a rat tibia. It was alsorecognized from the histopathological observation that there was inducedosteoconduction with the carbonate apatite block produced.

Comparative Example 4

This example relates to the production of carbonate apatite foam bysintering process, which process falls outside the scope of the presentinvention, in order to demonstrate the advantageous effects of thepresent invention. The starting carbonate apatite powders weresynthesized by wet process: Five liters of a sodium hydrogencarbonatesolution, prepared by dissolving sodium hydrogencarbonate in an aqueoussolution of 0.6 moles of sodium hydrogenphosphate, and five liters of a1M calcium acetate aqueous solution were concurrently added dropwise tothree liters of ion-exchanged water kept at 80° C., while controllingthe pH of the ion-exchanged water to be 9.0 to 9.5. On completing thedropwise addition, the resultant was maintained at 80° C. for twelvehours for aging, followed by filtration and washing operations so as toremove Na⁺ ions. The thus obtained powders were dried at 110° C. fortwenty-four hours. The X-ray diffraction pattern and theFourier-transformed infrared spectrum of the powder product showed thatit was composed of carbonate apatite. It was also shown found bymeasurement with a CHN analyzer that the carbonate group content was 9%by weight.

The carbonate apatite powders produced were admixed with distilled waterat a ratio of 1:1 to prepare a suspension. Polyurethane foam(Bridgestone Corp.) was immersed in the suspension of carbonate apatitepowders, followed by drying. The thus prepared polyurethane foam withthe carbonate apatite powders adhered to the trabeculae thereof wascalcined at 900° C. for five hours by raising the temperature at therate of 1° C. per minute up to 400° C. and then at the rate of 5° C.,whereafter the calcined product was cooled. Although it was confirmedthat there was formed a foamed structure in the furnace, it collapsedupon removal from the furnace. It was thus evidenced that it is notpossible to produce a desired carbonate apatite foam. The results aresimilar even with a calcination temperature of 1000° C., 1100° C., 1200°C. or 1300° C.

Comparative Example 5

This example relates to the production of carbonate apatite foam bysintering process, which process falls outside the scope of the presentinvention, in order to demonstrate the advantageous effects of thepresent invention.

The carbonate apatite powders prepared in Comparative Example 3 wereadmixed with distilled water at a ratio of 1:1 to prepare a suspension.Polyurethane foam (Bridgestone Corp.) was immersed in the suspension ofcarbonate apatite powders, followed by drying. The thus preparedpolyurethane foam with the carbonate apatite powders adhered to thetrabeculae thereof was calcined at 1400° C. for five hours by raisingthe temperature at the rate of 1° C. per minute up to 400° C. and thenat the rate of 5° C. per minute, whereafter the calcined product wascooled. Although the product was fragile, removal from the furnace wasmanaged. The X-ray diffraction pattern and the Fourier-transformedinfrared spectrum of the product showed that it was composed ofhydroxyapatite, and it was also found that the product contained nocarbonate group by a CHN analyzer. No resorption cavities by osteoclastcells were observed, indicating that there was no induced bonereplacement with the bone substitute material produced.

Comparative Example 6

In order to demonstrate the advantageous effects of the presentinvention, the carbonate apatite powder produced in Comparative Example4 (having an average particle size of 1 μm or smaller) was implantedinto bone defect site formed in a rat tibia. In two days from theimplantation there was a clear indication of inflammation, i.e., theskin at the implantation site became swollen. On incising the skin,there was observed a humoral yellowish transparent effusion. There wereno carbonate apatite powder present in the bone defect site, and noindication of osteoconduction whatsoever.

1. A method of producing a bone substitute material in the form of ablock predominantly composed of carbonate apatite for medical use, whichcomprises the step of forming carbonate apatite by contacting a block ofcalcium compound with a phosphate-containing solution, wherein saidcalcium compound block contains substantially no powders such thatpowders with a diameter of 20 micrometers or smaller are less than 1.0%by weight of said calcium compound block, wherein at least one of saidcalcium compound block and said phosphate solution contains a carbonategroup, and wherein the method does not include any sintering step, andwherein the porous block of calcium compound is one prepared using anartificially synthesized calcium compound.
 2. (canceled)
 3. The methodof producing a bone substitute material for medical use as claimed byclaim 1, wherein the calcium compound block prepared using anartificially synthesized calcium compound is a foamed calcium compound.4. A bone substitute material produced by a method comprising: formingcarbonate apatite by contacting a block of calcium compound with aphosphate-containing solution, wherein said calcium compound blockcontains substantially no powders such that powders with a diameter of20 micrometers or smaller are less than 1.0% by weight of said calciumcompound block, wherein the porous block of calcium compound is oneprepared using an artificially synthesized calcium compound, wherein atleast one of said calcium compound block and said phosphate solutioncontains a carbonate group, and wherein the method does not include anysintering step, and wherein the material is predominantly composed ofcarbonate apatite.
 5. The method of producing a bone substitute materialfor medical use as claimed in claim 1, wherein the contacting of theblock of calcium compound with the phosphate-containing solutioncomprises immersing the block in the phosphate-containing solution. 6.The method of producing a bone substitute material for medical use asclaimed by claim 1, wherein said calcium compound block containssubstantially no powders such that powders with a diameter of 20micrometers or smaller are less than 0.8% by weight.
 7. The method ofproducing a bone substitute material of claim 1, wherein said calciumcompound block contains calcium sulfate.
 8. The method of producing abone substitute material of claim 7, wherein said phosphate solutioncontains a carbonate group.
 9. The method of producing a bone substitutematerial of claim 8, wherein said phosphate solution contains ammoniumcarbonate.
 10. The method of producing a bone substitute material ofclaim 7, wherein said calcium compound block also contains calciumcarbonate.
 11. The method of producing a bone substitute material ofclaim 1, wherein said calcium compound block is a tricalcium phosphateblock and wherein said phosphate solution contains a carbonate group.12. The method of producing a bone substitute material of claim 1,wherein said phosphate solution contains ammonium carbonate.
 13. A bonesubstitute material claimed in claim 4, wherein the calcium compoundblock prepared using an artificially synthesized calcium compound is afoamed calcium compound.